Constitutive expression of functional GABA(B) receptors in mIL-tsA58 cells requires both GABA(B(1)) and GABA(B(2)) genes.

Studies of gamma-aminobutyric acid (GABA)(B) receptor function in heterologous cell systems have suggested that expression of two distinct seven transmembrane G-protein coupled receptor subunits is necessary for receptor activation and signal transduction. Some results suggest that both receptor proteins must be inserted into the plasma membrane to create heterodimers; however, it is possible that subunit monomers or homodimers are functional in cells which constitutively express GABA(B) receptors. A new pituitary intermediate lobe melanotrope cell clone (mIL tsA58) has been isolated which constitutively expresses GABA(B), D(2) and corticotrophin releasing factor receptors. Here, we report on characterization of the GABA(B) receptors. Solution hybridization-nuclease protection assays reveal the presence of GABA(B(1)) and GABA(B(2)) transcripts. Western blots show GABA(B(1a)) and one of two GABA(B(2)) proteins. Addition of the GABA(B) agonist baclofen to cultured mIL-tsA58 (mIL) cells inhibits high voltage activated Ca(2+) channels, as measured by agonist-induced inhibition of the K(+)-depolarization-stimulated increase in Ca(2+) influx. CGP55845, a GABA(B) antagonist, blocks the response to baclofen. Knockdown of either GABA(B(1)) or GABA(B(2)) subunits with selective antisense oligodeoxynucleotides reduced GABA(B) protein levels and completely abolished the GABA(B) receptor response in the mIL cells. Taken together, these results indicate that functionally active GABA(B) receptors in mIL cells require the constitutive expression of both GABA(B) genes. This is a physiologic validation of results from recombinant overexpression in naive cells and shows that the mIL cell line is a useful model for studying GABA(B) receptor expression, regulation and function.

The anticonvulsant, antihyperalgesic agent gabapentin is an agonist at brain gamma-aminobutyric acid type B receptors negatively coupled to voltage-dependent calcium channels.

Gabapentin (Neurontin, Pfizer Global R & D) is a novel anticonvulsant, antihyperalgesic, and antinociceptive agent with a poorly understood mechanism of action. In this study, we show that gabapentin (EC50 2 microM) inhibited up to 70 to 80% of the total K+-evoked Ca2+ influx via voltage-dependent calcium channels (VD-CCs) in a mouse pituitary intermediate melanotrope clonal mIL-tsA58 (mIL) cell line. mIL cells endogenously express only gamma-aminobutyric acid type B (GABA(B)) gb1a-gb2 receptors. Moreover, activity of the agonist gabapentin was dose dependently and completely blocked with the GABA(B) antagonist CGP55845 and was nearly identical to the prototypic GABA(B) agonist baclofen in both extent and potency. Antisense knockdown of gb1a also completely blocked gabapentin activity, while gb1b antisense and control oligonucleotides had no effect, indicating that gabapentin inhibition of membrane Ca2+ mobilization in mIL cells was dependent on a functional GABA(B) (gb1a-gb2) heterodimer receptor. In addition, during combined whole cell recording and multiphoton Ca2+ imaging in hippocampal neurons in situ, gabapentin significantly inhibited in a dose-dependent manner subthreshold soma depolarizations and Ca2+ responses evoked by somatic current injection. Furthermore, gabapentin almost completely blocked Ca2+ action potentials and Ca2+ responses elicited by suprathreshold current injection. However, larger current injection overcame this inhibition of Ca2+ action potentials suggesting that gabapentin did not predominantly affect L-type Ca2+ channels. The depressant effect of gabapentin on Ca2+ responses was coupled to the activation of neuronal GABA(B) receptors since they were blocked by CGP55845, and baclofen produced similar effects. Thus gabapentin activation of neuronal GABA(B) gb1a-gb2 receptors negatively coupled to VD-CCs can be a potentially important therapeutic mechanism of action of gabapentin that may be linked to inhibition of neurotransmitter release in some systems.